Method to improve the stability and broaden the pH range of family G/11 xylanases

Abstract

The present invention relates to protein engineering, and concerns especially family G/11 xylanases, and genes encoding said enzymes. In specific, the invention concerns Trichoderma reesei XYNII gene, which codes for endo-1,4-β-xylanase (EC 3.2.1.8). The invention describes how site-directed mutagenesis can be used to improve the properties of an enzyme to match the industrial conditions where it is used. Protein engineering can be used to improve thermoactivity and thermostability of xylanases, as well as to broaden their pH range.

The invention claimed is: 
     
       1. A recombinant gene encoding a variant xylanase having increased thermo stability or pH-stability when compared to a corresponding wild-type enzyme, wherein said variant comprises at least 95% sequence identity over the full length amino acid sequence of SEQ ID NO: 13, said variant having amino acid substitutions at positions corresponding to amino acids 2 and 28 of SEQ ID NO: 13. 
     
     
       2. The recombinant gene according to  claim 1 , wherein said variant has a cysteine at positions 2 and 28. 
     
     
       3. The recombinant gene according to  claim 2 , further comprising an arginine at amino acid position 58 and an aspartic acid residue (+191D) at the C-terminus of the enzyme. 
     
     
       4. The recombinant gene according to  claim 3 , wherein the wild-type enzyme has the amino acid sequence of SEQ ID NO:13. 
     
     
       5. The recombinant gene of  claim 3 , comprising SEQ ID NO:13 modified to have T2C, T28C, K58R, and +191D amino acids. 
     
     
       6. A recombinant gene encoding a variant xylanase having increased thermostability or pH-stability when compared to corresponding wild-type enzyme, wherein said variant comprises at least a 95% sequence identity over the full length amino acid sequence of SEQ ID NO:13, said variant further comprising an aspartic acid (+191D) at the C terminus. 
     
     
       7. The recombinant gene according to  claim 6 , wherein said variant has an arginine at a position corresponding to position 58 of SEQ ID NO:13.